An affinity assay is a biochemical test that measures the presence or concentration of a substance, the analyte, in solutions that frequently contain a complex mixture of substances, e.g., biological liquids such as blood, saliva, serum or urine. Such assays are based on the ability of a given molecule or a specific part of the molecule, e.g., an antibody to bind with high specificity to one or a very limited group of other molecules. The specificity of the assay depends on the degree to which the analyte is able to bind to its specific binding partner to the exclusion of all other substances in the sample to be analyzed.
In addition to the need for specificity, a binding partner must be selected that has a sufficiently high affinity for the analyte to generate an accurate and reproducible measurement, i.e., a measurable signal. Historically, this was accomplished by measuring a change in some physical characteristic such as light scattering or changes in a refractive index. With modern instrumentation, such methods are again becoming increasingly popular. Nevertheless, most immunoassays today depend on the use of an analytical reagent for binding to the analyte, which is associated with a detectable label. A large variety of labels have been demonstrated including radioactive elements used in radioimmunoassays; enzymes; fluorescent, phosphorescent, and chemiluminescent dyes; latex and magnetic particles; dye crystallites, gold, silver, and selenium colloidal particles; metal chelates; coenzymes; electroactive groups; oligonucleotides, stable radicals, polymers and others. Such labels serve for detection and quantitation of binding events either after separating free and bound labeled reagents or by designing the system in such a way that a binding event affects a change in the signal produced by the label.
An affinity assay usually probes the interaction of peptides, proteins, oligonucleotides, oligosaccharides or small molecules, such as aptamers, with immobilized binding partners. Typical binding partners include proteins, which can be receptors, enzymes or antibodies, but also polypeptides and polyamino acids (e.g., a poly-His tag for nickel binding sites, poly-lysine for amide or Schiff base linkages, poly-cysteine for thioether linkages). For example, streptavidin based immobilization schemes have widely been employed to attach a biotinylated biological element to an assay surface.
Affinity assays can be further classified for example, as competitive and non-competitive assays. In a competitive affinity assay, the analyte in the biological sample competes with a labeled analyte analogue to bind with its partner. The amount of labeled analyte analogue bound to the partner is then measured. In this method, the response signal will be inversely related to the concentration of the analyte in the biological sample. This is because the greater the response signal, the less analyte was available in the biological sample to compete with the labeled analyte analogue.
In non-competitive affinity assays, also referred to as the “sandwich assays”, an analyte in the biological sample is bound to its partner, then a labeled reagent is bound to the analyte. The amount of labeled reagent on the site is then measured. Unlike the competitive method, the results of the non-competitive method, i.e., the sandwich assay, will be directly proportional to the concentration of the analyte in the biological sample. This is because labeled reagent will not bind if the analyte is not present in the biological sample.
Affinity assays and, in particular, immunoassays are widely used as diagnostic tools in bacterial, viral, endocrine and parasitic diseases, as well as to detect drugs of abuse and chronic disease states, one example being heart disease which can lead to a heart attack.
Various techniques such as radioimmunoassay, immunoperoxidase, and ELISA are presently used for affinity assays and especially immunoassays. However, radioimmunoassays have the disadvantage of requiring the use of dangerous and environmentally unsound reagents.
Furthermore, apart from the specificity, as mentioned above, the sensitivity is of critical importance to the assay performance. An assay's sensitivity can be defined by the ratio of the specific signal generated by the binding event compared to the background noise of the system.
Factors decreasing the signal to noise ratio (SNR) include non-specific binding of reagents such as an antibody to various components of the assay. Moreover, the activity of some endogenous components of the assay matrix that react with the reagents of the assay, e.g., an enzyme substrate tends to yield a “false” reaction product that interferes with the accurate detection of the “true” product formed by a labeled complex, e.g., a labeled antibody-antigen complex.
Typically, additives such as blocking reagents are added to the assay matrix to lower assay noise and reduce the false positive signals.